title: Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V.
abstract: BACKGROUND
  DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning.
  
  
  RESULTS
  A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions.
  
  
  CONCLUSIONS
  The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed.
authors.affiliation: Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, Room UHG E2-143 Universitätsstr, 25, Bielefeld 33615, Germany. kristian@syntbio.net.
authors.forename: Tobias
authors.initials: T
authors.lastname: Baumann
authors.forename: Katja M
authors.initials: KM
authors.lastname: Arndt
authors.forename: Kristian M
authors.initials: KM
authors.lastname: Müller
mesh_terms: D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D007288:Inosine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013234:Stem Cells; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; D007288:Inosine; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; D043224:Deoxyribonuclease (Pyrimidine Dimer); C012271:deoxyinosine